Bismuth-213 generator and uses thereof

ABSTRACT

The present invention provides a generator capable of producing therapeutic Bismuth-213 doses. Also disclosed are methods of preparing Bismuth-213-labeled compounds using such generator and applications of the labeled compounds.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/079,902 filed Mar. 30, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field ofradioimmunotherapy. More specifically, the present invention relates toa Bismuth-213 generator and clinical uses thereof.

2. Description of the Related Art

The therapeutic potential of the alpha-particle emitting radionuclide,²¹³Bi, in the treatment of single cell neoplastic disorders such asleukemias (Nikula et al., 1998 and Jurcic et al., 1997), lymphomas, andmicrometastatic neoplasms (McDevitt et al., 1996) and possibly othercancers and diseases, gives the construction of a stable and reliableradionuclide generator a high priority. Bismuth-213 (²¹³Bi) is adaughter radionuclide of ²²⁵Ac and the decay cascade is shown in FIG. 1(Chang, 1996). There are six predominant radionuclidic daughters of²²⁵Ac which are produced in the cascade to stable ²⁰⁹Bi and for each²²⁵Ac decay there are a number of alpha-particle and beta-particledisintegrations, all of rather high energy. The cumulative ²²⁵Ac dose toa small mass of a functionalized organic resin due to 25 mCi ²²⁵Ac issubstantial and will rapidly cause complete generator failure to occur.Furthermore, the continuous generation of radical species on the resinand in the generator eluate can lead to poor radiochemical labelingyields and poor recovery of labeled antibody product.

Currently published generator technology (Kaspersen et al., 1995 andGeerlings et al., 1993) is not adequate to prepare material for humanuse. In its published form, virtually no useable Bi-213 can be extractedfrom the generator when it is loaded with quantities of Ac-225 necessaryto achieve human dose levels. This is because the concentrated Ac-225damages the column material, causes breakdown and fusion of the columnmedia, rapid leakage of Ac-225 from the column as well as leakage ofother non-isotopic by-products, and ultimately prevents elution of thecolumn or subsequent labeling reactions. Hence, a dose of Bi-213suitable for human use could not be obtained from the generatordescribed by Kaspersen or Geerlings (Kaspersen et al., 1995 andGeerlings et al., 1993) despite repeated attempts and the aforementionedproblems were not anticipated based on their published methods.

The prior art is deficient in the lack of effective means of producingdoses of Bi-213 suitable for clinical labeling for human use. Thepresent invention fulfills this long-standing need and desire in theart.

SUMMARY OF THE INVENTION

The present invention is directed to a method for constructing andoperating a ²²⁵Ac/²¹³Bi generator capable of producing 25-100 mCi of²¹³Bi suitable for clinical antibody labeling. The generator has beendesigned to have an effective lifetime of several weeks, producing up tosix therapeutic doses of radionuclide per day. To date, 80 clinicaldoses have been prepared and injected into patients using the described²¹³Bi generator.

The present invention is also directed to methods of preparing²¹³Bi-labeled phamarceutical compounds using the ²²⁵Ac/²¹³Bi generatorand related applications of these labeled compounds.

In one embodiment of the present invention, there is provided a Bi-213generator comprising a first container containing ²²⁵Ac solution; asecond container; a column; a third container; and a valve, wherein thevalve connects the first container, second container and the column.Preferably, the Bi-213 generator is capable of producing from about 10mCi to about 100 mCi of Bismuth-213 radioinuclide.

In another embodiment of the present invention, there is provided amethod for preparing a Bismuth-213-labeled compound, comprising thesteps of: (a) eluting the generator with an elution buffer to obtain aneluate; (b) adding to the eluate with the compound to be labeled forreacting; (c) adding a quench solution to the reaction; and (d)purifying the solution from (c) to obtain a final product, whichcontains Bismuth-213-labeled compound. Preferably, the processing timefor completing all the steps is from about 10 minutes to about 25minutes. Representative bismuth-213-labeled compound include anantibody, a fragment of an antibody, a cytokine and a receptor ligand.

In still another embodiment of the present invention, there is provideda system for preparing a Bismuth-213-labeled compound, comprising afirst container; the Bi-213 generator; a reaction vial; a secondcontainer; a column (or a filter); and a third container to collectfinal product, which contains Bismuth-213-labeled compound.

In still yet another embodiment of the present invention, there isprovided a kit for preparing a Bismuth-213-labeled compound based on theabove disclosed system.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the Ac-225 decay cascade with associated particulate decaysand half-lives.

FIG. 2 shows the apparatus 1 for constructing the ²¹³Bi generator,comprising a syringe 2 containing 1.5 M HCl equilibrated resin, asyringe 3 containing ²²⁵Ac/1.5 M HCl solution, a generator column 4 with“catch plug”, a syringe 5 to apply a negative pressure for loading theresin onto the column and a 3-way stopcock 6.

FIG. 3 shows the percent of total theoretical ²¹³Bi eluted with varyingiodide concentration: 0.10 M [I⁻]/0.10 M [Cl⁻] (diamonds); 0.05 M[I⁻]/0.15 M [Cl⁻] (squares); 0.04 M [I⁻]/0.16 M [Cl⁻] (triangles); 0.03M [I⁻]/0.17 M [Cl⁻] (x); and 0.02 M [I⁻]/0.18 M [Cl⁻] (stars).

FIG. 4 shows log of the dose from 25 mCi ²²⁵Ac to 200 (diamonds), 20(squares), and 1 (triangles) mg of resin vs. time.

FIG. 5 shows log observed ²¹³Bi eluate activity (diamonds) and logcalculated ²¹³Bi activity (squares) vs. time.

FIG. 6 shows absorption spectra of triiodide ion derived from prolongedair oxidation of a 0.1 M NaI/HCl solution (diamonds); H₂O₂ oxidation ofa fresh 0.1 M HCl/0.1 M NaI solution (squares); elution of a fresh 0.1 MHCl/0.1 M NaI solution through a ²¹³Bi generator (triangles); and 0.1 MNaI solution (circles). All solutions were diluted 100-fold inmetal-free water for measurement.

FIG. 7A is the time line for standard antibody labeling and purificationprocedure. FIG. 7B is the time line for improved antibody labeling andpurification procedure.

FIG. 8 shows reaction rate at different temperatures (acetate bufferpH=4.5) demonstrating that running the reaction at 37° C. may avoid aloss of 10% of the drug activity due to decay compared to 22° C.

FIG. 9 shows reaction rate at different temperatures (citrate bufferpH=6) demonstrating a similar temperature dependent pattern of thereaction rate when citrate was used as buffer at pH=6.

FIG. 10 is a schematic drawing of the improved radionuclide labeling andpurification system 7, comprising a syringe 8 with HCl, NaI, ascorbicacid; ²²⁵Ac/²¹³Bi generator 4, a breakthrough purification cartridge 9,a reaction v-vial 10 with mAb and acetate, a syringe 11 with EDTA andHSA, an anion exchange disc filter (or a column) 12, and a syringe 13 tocollect final product.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “²¹³Bi generator” shall refer to a device usedto prepare Bismuth atoms in pure form for therapeutic uses.

As used herein, the term “catch plug” shall refer to a part of thegenerator, comprised of resin, used to prevent elution of resinbreakdown products and parent radionuclides.

As used herein, the term “porous polyethylene frits” shall refer toplastic filters that retain resin in the generator.

As used herein, the term “barbed reducing fittings” shall refer toplastic connectors used to attach tubing to the generator.

As used herein, the term “alpha particle emitting” shall refer to atomsthat are radioactive and emit radiations comprising alpha particle.

As used herein, the term “beta particle emitting” shall refer to atomsthat are radioactive and emit radiations comprising beta particles(electrons).

As used herein, the term “therapeutic dose” shall refer to a dose ofdrug emitting radiation between 1 mCi and 100 mCi.

As used herein, the term “effective lifetime” shall refer to the periodof time that the generator is able to yield pure, reactive Bismuth-213.

As used herein, the term “radionuclidic purity” shall refer to thepercentage of Bismuth-213 relative to other radionuclides (e.g. Ac-225).

As used herein, the term “iodide eluate chemistry” shall refer to thechemistry of I⁻ and its various oxidation/reduction species withBismuth-213 that cause the Bismuth-213 to elute from the generator andfurther react with chelate.

As used herein, the term “antioxidant” shall refer to a chemical thatreduces the quantity of damaging oxidizing elements within a solution ordevice.

As used herein, the term “quench solution” shall refer to a chelatespecies to separate any unreacted Bismuth-213 and facilitatepurifications.

As used herein, the term “size exclusion purification” shall refer tothe use of a molecular sieve resin to separate molecules based on theirweights (or sizes).

As used herein, the term “anion exchange purification” shall refer tothe use of resin to separate molecules based on their electric charge.

The present invention describes a radioinuclide Bi-213 generator and themethod for construction and operation of the radionuclide generatorcapable of initially producing 10-200 mCi of Bi-213 (from Actinium-225)suitable for ligand or antibody labeling. Such ligands or antibodies areuseful for therapy of cancers and other diseases where a pathologicalcell must be selectively killed. The generator has an effective lifetimeof several weeks, producing up to six therapeutic doses of isotope perday, the limiting factor being the Ac-225 activity level. Factors suchas radiation damage to the generator, metal-ion contamination, Ac-225breakthrough, and isotope dilution that had previously limitedfeasibility of this approach were addressed and the improvements weremade to overcome these problems described. Successful clinical use ofthe product in humans was described as an example.

The present invention is directed to a generator for producingclinically usable Bi-213 for labeling. There are four significantchanges to the method, providing significant advances: 1) the Ac-225 isdistributed within the entire column matrix, thereby reducing localradiation dose. As a consequence, destruction and fusion of the columndoes not occur; 2) Washing of the column is performed so as to not allowbuildup of species that damage the column and prevent labeling andbuildup of byproducts; 3) Anti-oxidants were added to the bismuthproduct before adding the reaction mixture, to decrease radiolyticdamage to the antibody or other suitable carrier ligand; and 4) Catchplugs (guards) to prevent Ac-225 leakage were added. As disclosedherein, the use of catch plugs on the generator of the present inventionand the use of radiation (free radical scavengers) to protect column anddrug are significant.

The present invention is also directed to methods of preparingBi-213-labeled phamarceutical compounds for therapeutic uses.

In one embodiment of the present invention, there is provided a Bi-213generator comprising a first container containing ²²⁵Ac solution; asecond container; a column; a third container; and a valve, wherein thevalve connects the first container, second container and the column. TheBi-213 generator is capable of producing from about 10 mCi to about 100mCi of Bismuth-213 radioinuclide. Preferably, the container is asyringe, or a suitable reservoir, or a delivery device. The valve is a3-way stopcock. Still preferably, the second container contains achromatographic medium and the third container applies a negativepressure for loading the medium onto the column. Specifically, thechromatographic medium is resin.

In another embodiment of the present invention, there is provided amethod for preparing a Bismuth-213-labeled compound, comprising thesteps of: (a) eluting the generator with an elution buffer to obtain aneluate; (b) adding to the eluate with the compound to be labeled forreacting; (c) adding a quench solution to the reaction; and (d)purifying the solution from (c) to obtain a final product, whichcontains Bismuth-213-labeled compound. Generally, the processing timefor completing all the steps is from about 10 minutes to about 30minutes and the compound reacts with ²¹³Bi eluate at a temperature equalto or higher than room temperature. Preferably, the compound is selectedfrom the group consisting of an antibody, a fragment of an antibody, acytokine and a receptor ligand.

In one embodiment, the elution buffer is selected from the groupconsisting of NaI/HCl solution and NaBr/HCl solution. Further, it ispreferable that the elution buffer is mixed with an antioxidant, such asl-ascorbic acid.

In another embodiment, the compound to be labeled is premixed with anneutralizing buffer selected from the group consisting of NH₄Ac, Nacitrate and NH₄ citrate.

In still another embodiment, the quench solution is selected from thegroup consisting of EDTA, DTPA, EDTMP, EDTA+HSA, DTPA+HSA, EDTMP+HSA anda chelate.

In yet another embodiment, the purification method is selected from thegroup consisting of size exclusion chromatography, anion exchangepurification, reverse phase chromatography and affinity chromatography.

In still another embodiment of the present invention, there is provideda system for preparing a Bismuth-213-labeled compound, comprising afirst container; the Bi-213 generator; a reaction vial; a secondcontainer; a column (or a filter); and a third container to collectfinal product, which contains Bismuth-213-labeled compound. Preferably,the system further comprises a cartridge appended to the exit end of thegenerator to reduce the leakage of ²²⁵Ac. In one preferred embodiment,the first syringe contains an elution buffer selected from the groupconsisting of NaI/HCl solution and NaBr/HCl solution. Furtherpreferably, the elution buffer is mixed with an antioxidant, such asl-ascorbic acid. In this embodiment, the compound to be labeled ispremixed with an neutralizing buffer selected from the group consistingof NH₄Ac, Na citrate and NH₄ citrate. The quench solution may be EDTA,DTPA, EDTMP, EDTA+HSA, DTPA+HSA, EDTMP+HSA and a chelate. Representativepurification methods include size exclusion chromatography, anionexchange purification, reverse phase chromatography and affinitychromatography.

In still yet another embodiment of the present invention, there isprovided a kit for preparing a Bismuth-213-labeled compound based on theabove disclosed system.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Generator Construction

Bismuth-213 was produced from a “no carrier added” ²²⁵Ac source (VanGeel et al., 1994; Van Geel, 1995; Boll et al., 1997) provided as thenitrate from the Institute for Transuranium Elements (Karlsruhe,Germany) or Oak Ridge National Laboratory (Oak Ridge, Tenn.). The ²²⁵Acwas bound to an AGMP-50 cation exchange resin (Geerlings et al., 1993)utilizing modifications (McDevitt et al., 1996 and Finn et al., 1997)that allow the generator to yield the maximum recoverable amount of²¹³Bi every 5 hours (secular equilibrium is established) over a periodof several weeks.

Approximately 20-28 mCi of ²²⁵Ac residue was received dry inside aquartz ampoule which was attached with {fraction (1/32)} inch diametertubing to a 5.5 cm×0.64 cm column of inert fluoropolymer tubing (i.d.0.40 cm) with barbed reducing fittings, porous polyethylene plugs (0.16cm thick), acid washed quartz glass wool, and approximately 220 mg ofdry AGMP-50 resin, 100-200 mesh, H⁺ form (BioRad Laboratories, Hercules,Calif.). The ampoule and column were initially disconnected, and theampoule fitted with two 3-way stopcocks. The residue in the quartzampoule was digested with 0.5 ml of 3 M Optima grade HCl (FisherScientific), added through one of the stopcocks. The other end of thequartz ampoule containing the ²²⁵Ac acid solution was opened to a secondsyringe allowing release of any pressure increase due to heat or gasgeneration. The 3 M acid was allowed to contact the residue in thequartz ampoule for 0.5 hour with mild agitation to completely dissolveall of the ²²⁵Ac. After 0.5 hour the syringe used to apply the 3 M HClsolution was exchanged for a syringe containing 0.5 ml of metal-freewater and the acidic actinium chloride solution then diluted to yield a1.5 M HCl solution. The resulting acidic solution of actinium waswithdrawn into a 5 ml syringe, the ampoule washed once with 1 ml 1.5 MHCl and the wash combined with the ²²⁵Ac solution. The ampoule andsyringe are disconnected and the syringe 3 with the ²²⁵Ac solutionattached to the apparatus 1 depicted in FIG. 2.

While the ²²⁵Ac residue was being dissolved in the HCl the resin in thecolumn 4 provided was washed and equilibrated with 10 ml of 1.5 M Optimagrade HCl. The barbed fitting and the quartz wool on one end of thecolumn 4 was then carefully removed and approximately 210 mg of theresin was removed by backwashing into a clean plastic dish with asolution of 1.5 M HCl and approximately 200 mg of the removed resin wasplaced into a clean 10 ml syringe 2 in 2 ml of 1.5 M Optima grade HClsolution. The 10 mg of washed resin remaining in the column 4 was fixedin place with a small piece of acid washed quartz glass wool and willserve as a “catch plug” while the other 10 mg of resin that was setaside will eventually be loaded back in the column 4 and serve as second“catch plug”. These 10 mg sections of resin will serve as resin barriersto potentially capture ²²⁵Ac that might break through during routineelution. A larger independent “catch plug”, consisting of approximately100 mg of AGMP-50 resin, placed inside a column with porous polyethylenefrits and barbed reducing fittings, was later appended to the exit endof the generator to further reduce ²²⁵Ac breakthrough during routineoperation.

The partially assembled generator column 4, the syringe 3 with the ²²⁵Acsolution, and the 10 ml syringe 2 containing the 200 mg of the removedresin in 2 ml of 1.5 M Optima grade HCl solution were all attached via a3-way plastic stopcock 6. The exit end of the column was attached to a60 ml syringe 5 which was used to apply a negative pressure whilefilling the column 4. FIG. 2 illustrates the loading apparatus 1configured with all pieces in place.

Manipulation of the 3-way stopcock allows the acidic ²²⁵Ac solution tobe pulled into the syringe containing the acidic AGMP-50 resin slurry.This ²²⁵Ac solution and resin slurry are allowed to contact for 30 minwith occasional gentle agitation. After batch loading the ²²⁵Ac onto theresin support, the 3-way stopcock was again manipulated to load the²²⁵Ac/resin into the column. Loading was accomplished by positioning theapparatus so that the column now stands vertically to allow the²²⁵Ac/resin to flow downward. The resin was gently slurried prior topulling it out of the 10 ml syringe with a slight negative pressure topack the column. The stopcock position originally used for the syringe 3with the ²²⁵Ac solution was now attached to a clean syringe 2 with 10 mlof 1.5 M Optima grade HCl washing solution. This washing solution waspulled up into the 10 ml syringe (used to batch load the resin),agitated slightly to rinse the syringe, and then added to the generatorcolumn. The 60 ml syringe 5 was used to collect the wash solutionpassing through the column 4. The column 4 was disconnected from the3-way stopcock 6 and a small plug of acid washed quartz wool was appliedon top of the actinium loaded resin, followed by the remaining small 10mg “catch plug” of AGMP-50 resin and another small plug of acid washedquartz wool. A barbed reducing fitting was attached and the generatorwas ready to use. It is recommended to position the generator verticallyduring operation so that any fines produced would settle out and notembed into the polyethylene frits. The generator was then washed with 2ml of 1 mM metal-free HCl solution.

Actinium-225 activity received as a residue in a v-vial was dissolved in3 M HCl, allowed to digest, diluted with metal-free water, and batchloaded onto 1.5 M HCl equilibrated AGMP-50 and a generator constructedin the manner as described above. Metal-free reagents, plasticware, andapparatus were employed to construct and assemble the generator in orderto minimize the introduction of trace metals. The described methodallows the ²²⁵Ac activity to be handled in essentially a closed systemunder controlled conditions.

EXAMPLE 2 Radionuclide Detection and Quantification

Bismuth-213 activity was routinely measured with a Squibb CRC-17Radioisotope Calibrator (or equivalent model) (E.R. Squibb and Sons,Inc., Princeton, N.J.) set at 775 and multiplying the displayed activityvalue by 10. The activity value reported using the CRC calibrator wasverified by counting a 0.005-0.020 ml (±0.00015 ml) sample aliquot pointsource taken from an accurately known volume (2 to 10 ml±0.02 ml) ofgenerator eluate at a fixed geometry using a HPGe detector with pulseheight multi-channel analysis (Canberra Industries, Meriden, Conn.). TheHPGe detector counting efficiency of the ²¹³Bi 440 KeV g-emission wasdetermined for the same geometry from a plot of counting efficiency⁻¹vs. the γ-energy of standard radionuclide sources. The radioisotope dosecalibrator setting of 775 (multiplying the displayed activity value by10) was selected based upon measurements taken from the pulse heightmulti-channel analyzed g-emission spectrum of a ²¹³Bi sample. The ²¹³Bisamples, ranging in volume from 0.05 to 10 ml, were positioned at thebottom and center of the well of the CRC-17 Radioisotope Calibrator.

Pulse height multi-channel analysis and gas ionization detection (Ambis4000, Ambis Inc., San Diego, Calif.) were employed to determine theradionuclidic purity of the ²¹³Bi eluate and of the purifiedradiolabeled antibody construct, [²¹³Bi]Bi-CHX-A-DTPA-HuM195, by lookingfor ²²⁵Ac breakthrough. An aliquot of the generator eluate and/or finalproduct would be counted at least 75-100 hours post elution, allowingsufficient time for the original aliquot of ²¹³Bi activity to decay andfor the corresponding ²⁰⁹Pb activity (arising directly from the ²¹³Bialiquot) to also grow in and decay, leaving only the ²²⁵Ac breakthroughfor measurement. If there was breakthrough of ²²⁵Ac and daughters inequilibrium into the sample, then it would generate ²¹³Bi via secularequilibrium which could then be detected and quantified in theseanalyses. A standard curve was constructed for the ²²⁵Ac breakthroughanalyses by spotting serial diluted amounts of ²²⁵Ac in 1 M HCl andcounting these point sources on the Ambis 4000, using a standardizedgeometry, and counting the same sources on the multi-channel analyzer,using the 440 KeV ²¹³Bi emission to back calculate the amount of ²²⁵Acin equilibrium with the ²¹³Bi. Selected samples containing ²²⁵Acbreakthrough were held and counted repeatedly over a several week periodand the breakthrough activity decay values analyzed by curve fitting todetermine the identity of the radionuclidic breakthrough.

After generator construction the dispersion of the ²²⁵Ac on the resinsupport was verified by placing a freshly (<1 minute) eluted and washedgenerator on a piece of Type 52, 4×5 sheet film (Polaroid, USA) for anappropriate length of time and developing (approx. 5 min. exposure for a20 mCi generator). The exposed photographic image clearly shows theuniform dispersion of the radioactivity throughout the resin.

EXAMPLE 3 Generator Elution and Washing

A 0.1 M HCl/0.1 M NaI solution (Atcher, 1988) was prepared fresh eachtime and used to elute ²¹³Bi from the resin at a rate of approximately 1ml/min. The ²¹³Bi was eluted presumably as the (BiI₅)²⁻ anion species(Spivakov, 1979) with 0.1 M HI. Varying the iodide concentration (FIG.3) affects the elution characteristics of ²¹³Bi species from thegenerator. The ²¹³Bi reaches secular equilibrium with the ²²⁵Ac afterapproximately 300 min. (6.5 ²¹³Bi t_(½)'s), however, after approximately150 min. 90% of the maximum ²¹³Bi activity is available for elution fromthe generator. Approximately 3 ml of 0.1 M HCl/0.1 M NaI solution wasrequired to elute all of the recoverable ²¹³Bi from the generator. Themedium energy ²¹³Bi 440 keV photon emission (28% abundance) was readilyattenuated with a minimum amount of lead shielding and allowed for safehandling of multiple millicurie amounts of ²¹³Bi in the labeling andpurification steps. The generator was washed with 2 ml of 1 mMmetal-free HCl solution following each elution. The first 0.5 ml of thewash solution can be added to the 3 ml of 0.1 M HCl/0.1 M NaI elutionsolution to augment the ²¹³Bi yield.

EXAMPLE 4 Antibody Construct Radiolabeling and Purification

A freshly prepared 0.1 M HCl/0.1 M NaI solution was colorless, howeverafter passage over the generator resin, it elutes as a yellow coloredsolution due to the production of I₃ ⁻ ion. The ²¹³Bi eluate wasdecolorized by the addition of enough demetalized l-ascorbic acidsolution to produce a 5 g/l solution. The l-ascorbic acid also preventssignificant loss of radiolabeled antibody during the purification overP6 gel (see below), acting as a radioprotecting agent. Addition of 0.25ml of 3 M ammonium acetate was required to buffer 3 ml of 0.1 M HCl/0.1M NaI ²¹³Bi eluate to pH 4-5. Humanized monoclonal antibody (mAb)construct (0.3 to 0.5 mg), CHX-A-DTPA-HuM195 (TSI WashingtonLaboratories, Bethesda, Md.), was added to the buffered, radioprotected²¹³Bi solution and incubated at room temperature for up to 10 min.Following this incubation, 0.02 ml of 10 mM EDTA solution was added toquench the reaction mixture (i.e., chelate any remaining reactive ²¹³Bispecies).

The radiolabeled antibody was purified from the low molecular weightradioactive impurities, l-ascorbic acid, and other labeling reagents byusing a 10DG desalting column containing P6 gel (BioRad Laboratories,Hercules, Calif.) as the stationary phase and using 1% HSA as the mobilephase. Collection of approximately 7.5 ml of column eluate yields thefinal formulated product and the purity is determined by ITLC-SG.

In conclusion, the labeling and purification procedure for the alphaemitting radiometal Bi-213 includes: elute generator with a freshlyprepared 3 ml 0.1 N NaI/HCl solution; add 0.25 ml 3 N NH4Ac and checkpH; add 0.1 ml 150 g/l l-ascorbic acid; add antibody and check pH; reactfor 8-10 minutes with gentle mixing at ambient temperature; add 20 μl of10 mM EDTA quench solution; aliquot quality control sample; load ontoprewash/equilibrated size exclusion purification column; elute with 10ml of 1% HSA; collect fraction of elution; adjust antibody and HSAconcentration to prepare patient dose and aliquot quality controlsamples from residual product.

EXAMPLE 5 ²¹³Bi-Labeled Antibody Product Analyses

The observed radiolabeling reaction efficiency was determined by instantthin layer chromatography (ITLC-SG) using a 0.001 ml aliquot of thereaction mixture applied to silica gel impregnated paper (Gelman ScienceInc., Ann Arbor, Mich.) (Nikula, 1995). The paper strips were developedusing two different mobile phases. Mobile phase I was 10 mM EDTA and IIwas 9% NaCl/10 mM NaOH. The Rf of the radiolabeled antibody was 0 andboth the free metal species and metal chelates have Rf of 1.0 in mobilephase I. In mobile phase II, the radiolabeled antibody and free metalspecies have Rf of ˜0 and the metal chelates have Rf of 1.0. The stripswere cut at Rf=0.5 and counted in a Packard Cobra g-counter (PackardInstrument Co., Inc., Meriden, Conn.) using a 340-540 KeV window orcounted intact using a System 400 Imaging Scanner (Bioscan Inc.,Washington, D.C.) or the Ambis 4000.

EXAMPLE 6 Immunoreactivity Determination

The viability of the purified ²¹³Bi labeled antibody was ascertained bydetermining the immunoreactivity of the ²¹³Bi labeled CHX-A-DTPA-HuM195construct as described (Nikula et al., 1995) by incubating 2 ng ofradiolabeled mAb in 0.030 ml total volume with a 20- to 30-fold excessof antigen (10×10⁶ CD33 positive AL67 or HL60 cells). These cells haveapproximately 10,000-20,000 CD33 positive binding sites per cell (Nikulaet al., 1995) and have the capacity to bind up to 90% of added HuM195.After a 30 minute incubation at 0° C., the cells were collected bycentrifugation and the supernatant containing unbound mAb was removed toa second set of cells and incubated (30 min.) with the same amount ofexcess antigen as in the first incubation at 0° C. Under theseconditions of large antigen excess in a small volume, the reaction goesto near completion in 60 minutes. The percentage immunoreactivity wascalculated as equal to (bound radiolabeled CHX-A-DTPA-HuM195 constructto cells #1 plus cells #2)/(total bound plus unbound radiolabeledCHX-A-DTPA-HuM195 construct) times 100. Specific binding in these assayswas confirmed by lack of binding of the radiolabeled CHX-A-DTPA-HuM195construct to CD33 negative RAJI or MOLT 4 cell lines. To avoidnonspecific and Fc receptor binding, the assays were performed in thepresence of 2% human serum (Caron et al., 1992).

EXAMPLE 7 Iodide Ion Chemistry

The absorption spectra of 0.1 M HCl/0.1 M NaI solutions were obtainedfollowing i) prolonged air oxidation at ambient temperature; ii)oxidation by the action of 0.09% H₂O₂; and iii) elution through a ²¹³Bigenerator. These yellow colored solutions were decolorized upon addingan aliquot of l-ascorbic acid (5 mg/ml final [l-ascorbic acid]). Allfour of the solutions were initially 0.1 M HCl/0.11 M NaI and werediluted 100-fold for spectrophotometric measurement.

Results and Discussion

Previous attempts to load high levels of the acidic ²²⁵Ac chloridesolution (15-25 mCi) directly onto a prewashed, prepacked column ofAGMP-50 resin resulted in the ²²⁵Ac being deposited in a very smalllayer at the very top of the resin as confirmed by a 4×5 sheet filmimage of the generator. This method of loading resulted in thecatastrophic failure of the generator within 1-2 days of construction.Generator failure was defined as excessive ²²⁵Ac breakthrough,catastrophic radiolytic damage to the resin (sintering) and to theplastic body of the column (cracking). The sintering of the resinresults in the production of particulate fines which block the flow ofsolution through the generator. Generators constructed in this mannercould not be used for clinical preparations of radiopharmaceuticals. Theeffects of radiation damage to ion exchange materials are welldocumented (Gangwer et al., 1977) and it was known that organic cationexchange resins are considerably degraded when the total absorbed dosesare greater than 1×10⁸ rads. The total energy emitted per decay of ²²⁵Acis 4.493×10⁻¹² J (28.08 MeV).

The radiation dose to the AGMP-50 resin due to ²²⁵Ac and theradionulidic daughters in its decay cascade is large because of the 10day half-life of the parent radionuclide. In practice, 15-25 mCi of²²⁵Ac loaded onto the small mass of resin (<2 mg) at the top of thecolumn utilizing routine loading methods was observed to rapidly destroythe viability of the generator components and the resin material.Calculations of the dose from 25 mCi of ²²⁵Ac and the predominantdaughter radionuclides to 200, 20, and 1 mg of resin confirm theseobservations and serve as a guide for successful generator construction.The results of such calculations are shown in FIG. 4 plotting the log ofthe ²²⁵Ac dose to varying amounts of resin vs. time. For example, over a10 day period the dose to 1 mg of a polymeric cation exchange resin from25 mCi of ²²⁵Ac is 1.8×10¹¹ rads while the dose from the same amount ofactivity dispersed over 200 mg of resin is 8.8×10⁸ rads. Thesecalculations demonstrate the necessity of loading the ²²⁵Ac over a largemass of resin, especially when the organic cation exchange resins areconsiderably degraded when exposed to total absorbed doses greater than1×10⁸ rads. Greater resistance to radiolytic damage would be imparted byusing inorganic resin supports (Gangwer et al., 1977 and Wu et al.,1996), however, these materials may alter the reactivity of thegenerator eluate by the introduction of advantageous metal-ions ornecessitate time consuming elution conditions.

A fundamental improvement that allows for clinical use of the generatoris the uniform distribution of the ²²⁵Ac throughout the resin mass.Batch loading the ²²⁵Ac in the manner described above has resulted in agenerator that will produce chemically reactive ²¹³Bi for several weeks.The construction method described was used to prepare 12 clinicalgenerators resulting in 94.2%±5.9% (n=12) of the total ²²⁵Ac activityreceived being loaded onto the AGMP-50 resin for clinical use; 3.1%±2.8%(n=12) of the ²²⁵Ac was lost in the resin loading syringe (residualparticles); 2.3%±3.6% (n=12) remained in the quartz ampoule used forshipping; and 0.3%±0.2% (n=12) of the ²²⁵Ac activity was found in thecollected 1.5 M HCl washes. These percentages were determined after²²⁵Ac secular equilibrium was established and the ²¹³Bi in the washsolutions was allowed to decay.

Gas ionization detection of the β- and γ-emissions in the ²²⁵Ac decaycascade was used to determine the level of ²²⁵Ac breakthrough above the50×10⁻¹² Ci limit of detection. Pulse height multi-channel analyses ofgenerator eluates and formulated products using a HPGe detector has a20×10⁻⁹ Ci limit of detection such that detection of the presence of²²⁵Ac breakthrough in any of the aliquots was not seen. There was a lowlevel of ²²⁵Ac breakthrough in the generator eluate which ranged from2.0×10⁻⁶/ml±0.7×10⁻⁶/ml (n=5) during week 2 to 5.7×10⁻⁶/ml±2.1×10⁻⁶/ml(n=4) during week 3. The purification of the radiolabeling reactionmixture via size exclusion chromatography reduced the ²²⁵Ac breakthroughvalue to 0.29±10⁻⁶/ml±0.11±10⁻⁶/ml (n=6). The ²²⁵Ac breakthrough isexpressed as the nCi of ²²⁵Ac per ml of the eluate (or final product)per mCi of ²²⁵Ac on the generator.

The generator eluate ²²⁵Ac breakthrough value was significantly reducedto 0.13×10⁻⁶/ml±0.02±10⁻⁶/ml (n=3) by the addition of an independent“catch plug” of 100 mg of AGMP-50 resin packed into a small column thatwas attached to the exit end of the generator. The most practicalsolution was to append this 100 mg “catch plug” at the exit end of thegenerator thus reducing the ²²⁵Ac breakthrough into the generator eluateprior to radiolabeling, and utilizing the size exclusion chromatographyto further reduce ²²⁵Ac breakthrough value to 0.06±10⁻⁶/ml.Additionally, since the ²²⁵Ac breakthrough was primarily retained on theindependent 100 mg “catch plug” of AGMP-50 and recovered, it proved tobe a reasonable manner to manage the long-lived ²²⁵Ac contaminant.

Actinium-225 (and the daughters) was determined to be the radionuclidethat was present in the breakthrough samples. Selected samplescontaining ²²⁵Ac/²¹³Bi generator breakthrough were held and countedrepeatedly over a several week period. The breakthrough activity decayvalues were analyzed by curve fitting routines and found to fit an ²²⁵Acdecay curve.

A second improvement that allows clinical applications of this method isthe addition of l-ascorbic acid (reaction concentration of approximately5 g/l) to prevent significant loss of radiolabeled antibody during thepurification over P6 gel. This loss i s presumably a consequence ofprotein denaturation. In the absence of this radioprotecting agent,recovery of [²¹³Bi]Bi-CHX-A-DTPA-HuM195 after passage over a 10DGdesalting size exclusion column containing P6 gel was 47±25% (n=13) witha significant amount (22%±18% (n=13)) of radioactivity remaining on thecolumn after sufficient washing. Addition of l-ascorbic acid to thereaction mixture (n=28) results in a 69%±6% recovery of[²¹³Bi]Bi-CHX-A-DTPA-HuM195 with only 1.5%±1.4% of radioactivityremaining on the column after washing. This agent significantly improvesboth the yield and reproducibility of the labeling and purificationprocesses thus allowing production of clinically useful amounts of²¹³Bi. It is likely that other radioprotecting agents could besubstituted for l-ascorbic acid.

The observed radiolabeling reaction efficiency was 78%±9% (n=57) asdetermined by ITLC-SG. Purification via size exclusion chromatographyyields a product of 98%±2% purity (n=57) as determined by ITLC-SG. Totalprocessing time (i.e., the time from the end of generator elution toformulation of product for injection) was 23 min.±3 min. (n=56). Thespecific activity of the [²¹³Bi]Bi-CHX-A-DTPA-HuM195 construct was 14.3Ci/g±3.9 Ci/g (n=57). These radiolabeling, purification, and analyticalconditions were applicable to all the other antibody constructs thatwere examined.

The labeling reaction was allowed to proceed for 10 min. at ambienttemperature with gentle mixing yielding 78%±9% (n=57) ²¹³Biincorporation into the mAb. Although longer reaction times (20 min.)result in slightly higher yields of [²¹³Bi]Bi-CHX-A-DTPA-HuM195 (up toapproximately 88%, under standard reaction conditions), more activitywas lost through decay (loss of 23% after 20 min.) than was gained inhigher yield (increase of 10%) (Nikula et al., 1998). The fraction of[²¹³Bi]Bi-CHX-A-DTPA-HuM195 construct that was found to beimmunoreactive was 86%±10% (n=35).

Francium-221 was also eluted with the ²¹³Bi as evidenced by thecharacteristic decay curve exhibited by the generator eluate for along-lived daughter, short-lived parent, no equilibrium situation(Friedlander et al., 1981). FIG. 5 is a decay plot of log observedactivity of ²¹³Bi eluate and log calculated ²¹³Bi activity vs. time.Francium-221 posseses a 4.9 minute half-life and this radionucliderapidly decays to ²¹³Bi over the course of the elution, reaction,processing and delivery time so that most of the ²²¹Fr was eliminated bythe time of administration. The decay of this ²²¹Fr contributesapproximately 7.4%±0.2% more ²¹³Bi activity in addition to the originalamount of ²¹³Bi activity originally eluted. Furthermore, extending thegenerator elution flow rate to approximately 0.3 ml/min. allows for thecollection of an additional few percent of ²¹³Bi activity due to therapid regrowth of the ²²¹Fr on the generator.

A freshly prepared 0.1 M HCl/0.1 M NaI solution remains colorless for ashort period of time at room temperature in the presence of air, buteventually will turn yellow due to oxidation of the I⁻ ion in acidicsolution to the I₃ ⁻ ion by dissolved O₂ (Sigalla and Herbo, 1957). Theelution of a fresh (colorless) acidic iodide solution through a multiplemillicurie ²¹³Bi generator also yields a yellow colored solution. Theabsorption spectrum of the I₃ ⁻ ion (Awtrey and Connick, 1951) wasobserved in both the acidic air oxidized iodide solution (FIG. 6,diamonds) and the generator eluate (FIG. 6, triangles). The oxidizingeffect of ionizing radiation on I⁻ ions is a known phenomenon (Murfin,1959). The I₃ ⁻ ion absorption spectrum could also be obtained by theaddition of a 0.09% H₂O₂ solution to a fresh (colorless) acidic iodidesolution (FIG. 6, squares). The addition of l-ascorbic acid (finalsolution concentration of approximately 5 g/l) decolorizes all of theseyellow solutions, presumably by reducing the I₃ ⁻ ion and shifting theequilibrium of the reaction towards the I⁻ ion.

Multiple generators were constructed and linked in series in an effortto demonstrate the feasibility of devising generators in excess of onehundred millicuries of ²²⁵Ac. There were no remarkable differences inelution and recovery of ²¹³Bi or breakthrough of ²²⁵Ac relative to thatof the standard sized generator. All indications point to the ability toconstruct generators in excess of 100 mCi as soon as this amount of²²⁵Ac radionuclide becomes available without alterations in theradiolabeling conditions for the monoclonal antibodyradiopharmaceuticals.

In conclusion, applying the principles outlined above allows theconstruction of large alpha-particle emitting radionuclide generators(e.g. 100 mCi generators) utilizing organic resin supports to prepare²¹³Bi radiolabeled antibody constructs for clinical application.Radioprotecting agents such as l-ascorbic acid are necessary to ensurereproducibly high recovery yields of radiolabeled product during thepurification step. Breakthrough and recovery of ²²⁵Ac can be readilymanaged by employing a n independent “catch plug” cartridge appended tothe exit end of the generator or alternatively, increasing the intrinsic“catch plug” associated with each generator.

EXAMPLE 9 An Improved Clinical Labeling Procedure for the Alpha EmittingRadiometal Bi-213

In the field of radioimmunotherapy, that is, treating human disease byuse of radioisotopes targeted to specific sites or tissues with animmune protein, antibody or small molecule, there is a need to have amethod to prepare short-lived, potent radiopharmaceuticals suitable forwidespread human use. This is particularly true in the field of alphaparticle radioimmunotherapy which uses Bi-213, an isotope with a 46minutes half-life. The processing time for above discussed labeling andsize exclusion purification method is more than 25 minutes (see FIG. 7Afor the time line for standard labeling procedure). Due to a 46-minhalf-life of Bi-213, there is significant decay during the 25 minutes ofprocessing, and 20-30% of drug is lost. Improvements to shorten theprocessing time while at the same time making a process that isreproducible and maintains the integrity of the generator and drugproduct is necessary for widespread use of the drug.

The labeling and purification procedure was modified with the followingfive improvements resulting in markedly reduced preparation time andincreased stability of the product: 1) combine antibody with 3 N NH4Acand directly elute Bi-213 into the antibody solution; 2) add l-ascorbicacid into 0.1 N NaI/HCl solution as eluate; 3) warm the generator andreaction tube under 37° C. and allow reaction for 2 minutes; 4) mix EDTA(or other chelates) and HSA as a quench solution; 5) anion exchangepurification. The processing time after implementing all themodifications was about 10 minutes (see FIG. 7B for the time line forimproved labeling procedure). Oxidation of the product and generator wasreduced as well. The improvements lead to reduced time of processing,and thus less decay of the drug activity. Less decay means more isotopeavailable per elution (dose), therefore more patients can be treated.The following describes the improvements and the experimental method foreach step:

Improvement #1: Preparing the antibody directly in the neutralizingbuffer, and assessing the stability of CHX-A-DTPA-HuM195 in 3 N NH4Ac.The goal is to mix monoclonal antibody with 3 N NH4Ac for long termstorage without loss of its reactivity and immunoreactivity. Suchmodification provides the advantage of simplifying labeling procedure bycombining at least two steps (adding NH4Ac and adding HuM195) into one,on which a simple kit for human use of Bismuth labeled antibodies can bebased.

In detail, CHX-A-DTPA-HuM195 (0.094 ml, 10.6 mg/ml) was added into 1 mlof 3 N NH4Ac and stored in a refrigerator (2-4° C.). Labeling tests wereperformed with three different radiometals on day 13 (two weeks), withBi-213; day 20 (three weeks), with Y-90; day 61 (two months) withIn-111. The results are summarized in Table 1. The results show thatCHX-A-DTPA-HuM195 in 3 N NH4Ac after two months still retains ability tochelate In-111 without losing its immunoreactivity. Hence, it ispossible to make a stable kit with all the ingredients needed for thereaction for storage.

TABLE 1 HuM195 Stability in 3N NH4Ac and Labeling Test with Bi-213,In-111, and Y-90 DTPA to Test Day Isotope Reaction Metal No After UsedCode Activity Antibody Ratio 1 13 Bi-213 R8.090398 5.14 mCi 0.228 mg1183 2 20 Y-90 R1.091098 7.64 mCi 0.228 mg 10 3 61 In-111 R2.102198 3.29mCi 0.228 mg 22 Reaction Test Yield Purified Product ImmunoReactivity NoITLC HPLC ITLC HPLC AL67 Control 1 85% 84% 96% ˜100% NA NA 2 85% 80% 96%˜100% 86.30% 3.5% LNCAP 3 87% 86% 98% ˜100% 96.2% 14.8% RAJI

Improvement #2: Elimination of oxidants by elution of Bi-213 from theAc-225 generator using a 0.1 N NaI/HCl mixed with l-Ascorbic acidsolution as eluent. Significant oxidation of the generator column andthe elution buffers occurs due to the extraordinary radiation flux. Upto 3 billion rad are generated in the column. Eluents are oxidized andyellowed upon retrieval from the column. One solution to this problem isto mix l-ascorbic acid (an antioxidant) with 0.1 N NaI/HCl solution forelution; there are concerns that complicated oxidation-reductionreactions could occur inside the column that would prevent routinefunctioning of the generator. However, addition of 100 ul of l-ascorbicacid (150 g/l, demetalized) into 3 ml of 0.1 N NaI/HCl as eluentdemonstrated appropriate elutions of Bi-213 (Table 2). A freshlyprepared 0.1 N NaI/HCl solution is colorless, however, after passageover the Ac-225 generator, it elutes as a yellow colored solution due tothe production of I₃ ^(⁻) ion. When the l-ascorbic acid step was added,colorless solutions were eluted off the generator indicating thatl-ascorbic acid acts as a reducing agent to keep iodine in the I⁻ formunder alpha particle irradiation. The test labeling of Bi-213 elutedusing l-ascorbic acid incorporated eluent with CHX-A-DTPA-HuM195 resultsin a 80% reaction yield which is the typical yield using only 0.1 NNaI/HCl as eluent and adding the l-ascorbic acid after elution andbuffering.

TABLE 2 Ac-225/Bi-213 Generator Elution Using 0.1N NaI/HCl Mixed withl-Ascorbic Acid Bi Bi Re- Preparation Generator Activity coveredPhysical Labeling Code Activity Eluted (%) Appearance Yield E8 5.10 2.3994 colorless NA (Dec. 7, 1998) E9 4.99 2.58 104 colorless NA (Dec. 8,1998) R10 4.52 2.37 105 colorless 80% (Dec. 9, 1998)

As mentioned above, a freshly prepared 0.1N NaI/HCl solution iscolorless. However, prolonged air oxidation of a 0.1N NaI/HCl solutionalso oxidizes and yellows. As a consequence, the generator needs to beeluted with a freshly prepared 0.1 N NaI/HCl (by mixing equal volumes of0.2 N NaI and 0.2 N HCl solutions). L-ascorbic acid will prevent 0.1 NNaI/HCl solution from yellowing for at least four weeks. Because theaddition of l-ascorbic acid can improve properties of NaI/HCl solutionfor a month, it is not necessary to prepare a fresh 0.1 N NaI/HCl foreach elution. This may allow the generator operation to be robotic inthe future.

Improvement #3: Markedly decrease reaction times by running the reactionat 37° C. compared to 22° C. The current published reaction method isconducted at room temperature for 8 to 10 minutes. Due to the shorthalf-life of Bi-213, this results in a significant loss of isotope. Ifthe reaction time can be shortened from 8-10 minutes to 1-3 minutes, itmay avoid a loss of 10% of the activity of the drug due to decay.

In detail, the Bi-213 solution was warmed up to ˜37° C. in a water bathor a heating unit and then CHX-A-DTPA-HuM195 was added for reaction. Thekinetics of the reaction was monitored by taking aliquots of thereaction sample at different times. The reaction yield was determinedusing the ITLC (EDTA) method. The reaction yield vs. time was plottedcompared to the reaction at room temperature (FIG. 8). The earliest datapoint taken was at 1 min. It was shown that the reaction went tocompletion at 1 min at 37° C. with ˜85% yield and at 5-7 min at roomtemperature with ˜82% yield. Although increasing the temperature doesnot increase the reaction yield, the reaction rate has beensignificantly accelerated. A similar temperature dependent pattern ofthe reaction rate was observed when citrate was used as buffer at pH=6(FIG. 9).

As mentioned above, the experiment was conducted at 37° C. by preheatinga Bi-213 solution to 37° C. and then adding antibody construct forreaction. However, the same reaction conditions may not allow preheatingof the Bi-213 solution after elution. Therefore, other possibilities areto heat up either the 0.1 N NaI/HCl/l-ascorbic acid solution beforeelution or Bi-213-NaI/HCl/l-ascorbic acid solution after elution. Threeoptions have been tested to learn the efficiency of the heating: 1) NaIand HCl solutions heated separately in a water bath or a heating unitwere mixed and then used to elute the column; 2) the eluent was heatedby embedding the tubing between the column and the external “catch”column in a water bath; and 3) the two methods were combined. A reactiontube with a thermometer inside was placed in a water bath and used tocollect eluent. Heating the column and the reaction tube together isanother possibility.

Improvement #4: Shorten reaction times by using EDTA mixed with HSA as aquench solution. An EDTA solution was added to chelate any reactive freeBi-213 and Bi-213 which was non-specifically bound on the antibody. Freeradiometal may be extremely dangerous upon infusion into the patient.Anion exchange purification (discussed later) will be used to separateBi-213-EDTA from the radiometal chelated conjugated antibody solution.Anion exchange purification requires mixing HSA with quenched Bi-213antibody solution before loading onto the column.

It was investigated whether EDTA can be mixed with HSA as a quenchsolution. Since HSA solution contains some metal ions (such as Fe, Zn,and Cu), it will consume EDTA. Hence, the question is how much is EDTAneeded to accomplish its function as a chasing agent. Differentconcentrations of EDTA in HSA solutions have been prepared by adding 20μl of 10 mM EDTA, 100 μl of 10 mM EDTA, and 100 μl of 100 mM EDTA into 1ml of 5% HSA to make 0.2, 2, and 20 mM EDTA-HSA solutions, respectively.Indium-111 in 0.05 M HCl (NEN) was buffered to pH 4.5 using 3 N NH4Ac.The buffered In-111 solution was mixed with EDTA-HSA solutionscontaining 0, 0.2, 2, 20 mM EDTA. Aliquots of each sample, includingbuffered In-111 stock solution, were analyzed using the ITLC method todetermine the percentage of free In-Ill and In-111-EDTA complex. Theresults are shown in Table 3.

TABLE 3 Mixing EDTA and HSA as a Quench Solution Exp. In-111 EDTA In-111at origin No Mixed with Concentration ITLC (NaOH) 1 Nothing 0 mM 95 2HSA 0 mM 95 3 EDTA-HSA 0.2 mM 0 4 EDTA-HSA 2 mM 0.3 5 EDTA-HSA 20 mM 0

It shows that without EDTA (Exp. 1 and 2), 95% of In-111 remainedimmobile at the origin in NaOH-ITLC. However, most of In-111 moved atthe solvent front when EDTA added into HSA at all three concentrationsof EDTA (from 0.2 to 20 mM, Exp. 3, 4, and 5). This indicates that EDTAeven at 0.2 mM concentration can still efficiently chelate In-111 inpresent of 5% HSA.

Improvement #5: Rapid separation of the drug product by anion exchangepurification. The current size exclusion purification method takes 6 to10 minutes, which accounts for a 15 % loss of Bi-213 activity due todecay. A simple and fast anion exchange purification technique has beendeveloped to reduce the purification time and simplify the operation.The objective is to convert Bi-213 impurities into anionic species,which can be quantitatively retained on an anion exchange column, and torefine conditions to allow antibody construct to pass through the columnquantitatively without denaturing the IgG. When quenched Bi-213 antibodysolution is mixed with human serum albumin (HSA, 1-5%) in isotonicsaline, the AG1X8 resin in acetate form adsorbs the Bi-213 impurity andallows quantitative Bi-213 antibody elution (Table 4).

TABLE 4 Comparison of Different Concentrations of HSA Activity ProductAntibody Impurity HSA (%) Eluted (%) Purity (%) Recovery (%) Eluted (%)0.5 63.3 95.9 76.3 2.6 2.5 71.7 96.3 86.8 2.6 0.5 wash added 74.5 97.090.8 2.2 2.5 wash added 81.9 96.5 99.3 2.8

The reaction was quenched with DTPA and yield was 80%. Sample of 3 ml(1.5 ml ²¹³Bi+1.5 ml HSA) was loaded onto the column and collected (rows1 and 2) and followed with a 3 ml wash (rows 3 and 4).

Thus, a rapid anion exchange purification method can be developed andcan provide immunoreactive Bi-213 labeled antibody product with >95%purity and >95% antibody recovery. It was also demonstrated that HSAwashes of the anion exchange column yields higher recovery. Theprocedure can be accomplished in less than 3 minutes. The technique canbe reproducibly used by non-technical staff.

In conclusion, the improved radionuclide labeling and purificationsystem 7 comprises a syringe 8 with HCl, NaI, ascorbic acid; ²²⁵Ac/²¹³Bigenerator column 4, a breakthrough purification cartridge 9, a reactionv-vial 10 with mAb and acetate, a syringe 11 with EDTA and HSA, an anionexchange disc filter (or a column) 12, and a syringe 13 to collect finalproduct (FIG. 10). This system can also be used for labeling andpurifying non generator produced radionuclides, e.g., Y-90, In-111, etc.

EXAMPLE 10 Clinical Use of Generator Produced 213-Bismuth HuM195 Ligand

A clinical experiment (protocol) describing one possible use of an alphaemitting targeted construct follows. This experiment describes the useof the HuM195 IgG1 to target Bismuth-213 to leukemia cells anddemonstrates that the constructs are stable, will deliver the isotope tothe cells in a human, and will kill leukemia cells without apparenttoxicity to non target tissues. Such a scheme might be used with anotheralpha emitter, such as Bismuth-212 attached stably to this or anotherligand, such as an antibody or fragment, cytokine, or receptor ligand,each of which is capable of specific and high affinity binding to atarget cell or tissue. Moreover, such ligands might be used toselectively kill nonmalignant targets such as lymphoid cell involved ina pathological process such as inflammation or autoimmunity.

Acute myelogenous leukemia (AML) is the predominant type of acuteleukemia in adults. While most patients are able to achieve a completeremission with chemotherapy consisting of cytosine arabinoside and ananthracycline, prolonged disease-free survival is less than 20%.Reinduction attempts will produce second remissions in only 20-25% ofpatients, frequently lasting less than 6 months. Less than 5% ofrelapsed patients will survive one year (Stone et al., 1993).

Chronic myeloid leukemia (CML) is a biphasic disorder of earlyhematopoietic progenitors. The chronic phase (with a median duration of4 years) is associated with marked elevations of mature and maturingleukocytes and leads invariably to a blastic phase resembling acuteleukemia. Treatment with α-interferon has been shown to eradicateevidence of the Philadelphia chromosome by cytogenetic analysis in aminority of patients. Treatment with conventional chemotherapy, however,has had no impact on the natural history of this disease. Allogeneicbone marrow transplantation is the only potentially curative option forthese patients. Since patients in accelerated or blastic phases of CMLgenerally do not benefit from transplantation, efforts have been made totransplant these patients during the early chronic phase of theirdisease (Kantarjian et al., 1993).

Classified as a myelodysplastic syndrome, chronic myelomonocyticleukemia (CMMOL) is defined by the presence of a monocyte count ofgreater than 1 H 109/1, monocytosis of the bone marrow, anemia, andthrombocytopenia. Survival ranges from several weeks to years, with amedian survival of 30 to 41 months. Treatment is mostly palliative;hydroxyurea can be used to control high peripheral blood leukocytecounts (List et al., 1990)

HuM195: A fully humanized M195 construct that has improved biochemicaland immunological activities. Complementarity-determining region(CDR)-grafted humanized M195, retaining only the CDRs and othersterically important amino acids from mouse M195 were constructed usinghuman IgG1 frameworks. Sp2/0 mouse myeloma cell lines secretinghumanized M195 were grown in vitro and the antibodies were purified onPA-Sepharose by affinity chromatography using sequential pH stepelutions. Purity was determined on SDS-polyacrylamide gels stained withCoomassie brilliant blue (Co et al., 1992).

The humanized M195 (HuM195) construct maintained binding specificityconfirmed against a panel of CD33+ and CD33− cell lines byradioimmunoassays. Specificity for fresh hematopoietic samples from 47patients was confirmed using a direct fluorescein conjugate of HuM195.Direct radiobinding assays showed the ability of HuM195 to beinternalized rapidly after binding to the cell surface. HuM195 showed upto an 8.6-fold higher binding avidity than the original mouse M195.HuM195 is effective in inducing rabbit complement-mediated cytotoxicityagainst HL60 cells, and fibroblasts transfected with CD33 genes. Unlikemouse M195, HuM195 demonstrated the ability to induce antibody-dependentcell-mediated cytotoxicity (ADCC) using human peripheral bloodmononuclear cells as effectors. ADCC was enhanced by the addition ofIL-2 (Caron et al., 1993).

With the use of HuM195, the problem of immunogenicity can largely beeliminated. Potentially, unconjugated HuM195 may have activity in thesetting of minimal residual disease where effector cell populations arepresent, and clinical trials are now underway to evaluate the utility ofthis agent in the post-remission setting. Unconjugated HuM195, however,is unlikely to have significant activity in patients with overt leukemiabecause of the lack of effector cells. Therefore, a less toxic M195construct that does not require marrow transplantation is warranted.

Alpha particle emitting constructs of HuM195: Due to the high linearenergy transfer and short (10-80 μm) range of alpha particles, alphaemitter-labeled antibodies are highly efficient in killing tumor cellsor small tumor cell clusters, but no monoclonal antibodies (mAbs)labeled with alpha-particle emitters have been used in humans. Bismuthhas two potentially useful alpha-emitting isotopes, ²¹²Bi and ²¹³Bi,which have half-lives of 61 and 46 minutes, respectively. Both ²¹²Bi and²¹³Bi-generator systems yield high purity isotopes capable of chelationto monoclonal antibodies. In an experimental system, approximately 25cell-bound α-particle-emitting immunoconjugates per target cell and fourα-particle traversals through the nucleus were required to reduceclonogenic survival of a lymphoma cell line by 90% (Macklis et al.,1992). Specific cytotoxicity of ²¹²Bi-labeled anti-Tac (anti-CD25)monoclonal antibody has been demonstrated against an adult T-cellleukemia line in vitro (Kozak et al., 1986). In previous studies, miceinoculated intraperitoneally with the murine tumor line EL-4 were curedof their ascites after intraperitoneal injection of 150 μCi of a²¹²Bi-labeled antibody conjugate. Animals receiving over 400 μCi of²¹²Bi showed signs of radiation exposure such as weight loss, diarrhea,and infection (Macklis et al., 1988). Additionally, in vivo specificityand efficacy of radioimmunotherapy with ²¹²Bi and ²¹¹At have been seenin murine erythroleukemia and neoplastic meningitis models, respectively(Huneke et al., 1992)

Alpha emitters have now been conjugated via a bifunctional chelate(CHX-A-DTPA) to HuM195 with high efficiency (>90%) and high specificactivities (up to 20 mCi/mg). This chelator had a high avidity forradiometals and lack of immunogenic responses in experimental animals(Camera et al., 1994). ¹¹¹In-, ²⁰⁶Bi-, ²¹²Bi-, and ²¹³Bi-HuM195constructs have been made. The immunoreactivity of chelated HuM 195 (HuM195-CHX-A-DTPA) varied from 80 to 95%. HuM195-CHX-A-DTPA was rapidlyinternalized into cells. In vitro cell killing experiments withdifferent specific activities of ²¹²Bi-HuM195 showed dose-dependent andspecific activity-dependent killing of HL60 target leukemia cells.²¹³Bi-HuM195 was injected into healthy BALB/C mice at doses of 0.5 to 20mCi/kg. No changes in weight, viability, hemoglobin, or leukocyte countwere seen. At doses of 70 mCi/kg, two of three mice died within twoweeks and a third showed significant reductions in leukocyte counts. Thedoses planned in this clinical trial range from 0.28 to 0.7 mCi/kg.Mathematical modeling predicted the red marrow dose from ²¹³Bi to be0.55 mGy/MBq or 1.5 mSv/MBq. The percentage of tumor cells killed for 10gm or less of single isolated cells was >99.9% and approximately 50% for100 gm of disease when 370 MBq (10 mCi) of ²¹³Bi were administered.

Clinical grade ²¹³Bi generators capable of producing 25-50 mCi will beprepared. Actinium is supplied dried onto a glass ampule from theTransuranium Elements Institute in Karlsruhe, Germany. ²¹³Bi is elutedfrom the generator and chelated to HuM195-CHX-A-DTPA, followed byseparation of ²¹³Bi-HuM195 by size exclusion chromatography. Sensors forOD280 and (emissions will be used to determine yield and specificactivity. Unlabeled HuM195 may be added to adjust the dose as necessary.This will be performed at Sloan-Kettering Institute immediately prior toinjection into patients. ²¹³Bi-HuM195 will be diluted in normal salinewith 1% human serum albumin (HSA) to a total volume of 10 ml forinjection.

Patients will be treated in the Nuclear Medicine Department on either anoutpatient or inpatient basis. Given the short-half life of ²¹³Bi,radiation exposure to hospital staff will be minimal and radiationisolation for patients is not required. Patients will be monitored by aRadiation Safety Officer and instructed in the proper disposal of waste.Additionally, patients will not be discharged for two to three hoursafter infusion, by which time any remaining gamma radiation will betrivial. Patients will receive ²¹³Bi-Hum195 by IV push over 5 minutestwice daily, 5-8 hours apart, according to the dose escalation schemebelow:

Dose Level 1 (0.28 mCi/kg):

Day 1: 0.07 mCi/kg BID

Day 2: 0.07 mCi/kg BID

Dose Level 2 (0.42 mCi/kg):

Day 1: 0.07 mCi/kg BID

Day 2: 0.07 mCi/kg BID

Day 3: 0.07 mCi/kg BID

Dose Level 3 (0.56 mCi/kg):

(4 days as above)

Dose Level 4 (0.7 mCi/kg):

(5 days as above)

Because of the short half-life of ²¹³Bi, individual doses of ²¹³Bi mayvary by 20%. Efforts will be made to keep the cumulative dose as plannedby adjusting subsequent doses. At least three patients will be treatedat each dose level according to the dose escalation scheme. Ifacceptable toxicity is encountered, patients at the next dose level willbe enrolled.

Eleven patients have entered on four dose levels. More than 50 doses ofthe drug were synthesized according to the specifications and injectedinto the patients. Doses could be prepared from the generator at leastevery 3-4 hours. The generator provided drug that met specifications forat least nine days allowing the treatment of patients. Real timepharmacokinetics were assessed by gamma imaging and serial blood work.The drug targeted first to the sites of leukemia and monocyte/macrophagecells in the liver and spleen. The bone marrow was targeted next. Overtime, with succeeding injections, uptake into the liver decreased by 50%as sites were saturated, and uptake in the bone marrow increased by100%, as more drug became available. The estimated radiation doses inREM to the whole body, kidneys or other non-target organs were 0.03; tothe blood, 125; to the liver 600; to the spleen 1400;1 and to the redmarrow, 1100. Target to non-target dose ratios were therefore25,0000-50,000 to one. There was no acute toxicity in any patient. Therewas no extramedullary toxicity seen in any patient. In most patients,peripheral blood cell counts (leukemia blasts and white cells) began tofall within 48 hours after treatment and were reduced by up to 90%.Counts usually returned within two weeks. The bone marrow at one weekshowed reduced cellularity and reduced leukemia blast percentages in themajority of patients (up to 70% reduced).

Conclusion from initial levels: The drug can be prepared andadministered safely and repeatedly without extramedullary toxicity. Thedrug displayed pharmacokinetics consistent with rapid, specific, andstable targeting only to appropriate cancer cell sites. Significantanti-leukemic activity was seen even at the lowest level. Doseescalation will continue.

In conclusion, a generator capable of producing multiple high levels ofBismuth for labeling of clinically useful ligands (or antibodies) isdescribed. This generator conforms to the essential physicalrequirements (yield of product and radiochemical and radionuclidicpurity) outlined in (Knapp et al., 1984 ).

The following references may have been cited herein:

Atcher, et al., (1988) Appl. Radiat. Isot. 39, 283.

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Huneke, et al., (1992) Cancer Res 52: 5818-5820.

Jurcic, et al., (1997) Blood 90(suppl), 504a.

Knapp, et al., eds, Radionuclide Generators, ACS symposium Series #241,ACS, Washington, D.C., 1984, p185.

Kozak, et al., (1986) Proc Natl Acad Sci USA 83:474-478.

Macklis, et al., (1992) Radiation Res 130: 220-226.

Macklis, et al. 91988) Science 240:1024-1026.

McDevitt, et al., (1996) Tumor Targeting 2(3), 182.

Murfin,(1959) J. Inst. Sci. Techn. 5(4), 10.

Nikula, et al., (1995) Nucl. Med. Biol. 22, 387.

Nikula, et al., (1998) J. Nucl. Med. (to be published)

Sigalla, et al., (1957) J. Chim. Phys. 54, 733.

Spivakov, et al., (1979) J. Inorg. Nucl. Chem. 41, 453.

Van Geel, et al., (1994) European Patent nb. 0 443 479 B1.

Van Geel, (1995) ITU Annual Report 1995-(EUR 16368)-Basic ActinideResearch 55.

Wu, et al., (1996) Abstracts of Papers of the American Chemical Society212(1), 61-NUCL.

Zalutsky, et al., (1994) Cancer Res 54: 4719-4725.24.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

What is claimed is:
 1. A bismuth-213 generator, comprising: a first container containing ²²⁵Ac solution; a second container; a generator column; a valve, said valve simultaneously and directly connected to an end of each of said first container, said second container and said column; and a third container, said third container connected to said generator column at an exit end opposite to the end connected to said valve.
 2. The generator of claim 1, wherein said first container, said second container and said third container are individually a syringe or a delivery device.
 3. The generator of claim 1, wherein said valve is a 3-way stopcock.
 4. The generator of claim 1, wherein said second container contains a chromatographic medium, and said third container applies a negative pressure for loading said medium onto said generator column.
 5. The generator of claim 4, wherein said chromatographic medium is resin.
 6. The generator of claim 5, wherein said resin is equilibrated with HCl solution.
 7. The generator of claim 1, further comprising a cartridge appended to the exit end of said generator column between said exit end and said third container.
 8. The generator of claim 7, wherein said cartridge reduces the breakthrough of ²²⁵Ac actinium-225 eluted from said generator column.
 9. A method for preparing a bismuth-213-labeled compound, comprising the steps of: (a) operating the bismuth-213 generator of claim 1 to load actinium-225 throughout a resin in the generator column; (b) eluting the generator column with an elution buffer to obtain an eluate; (c) adding said compound to said eluate to react; (d) adding a quench solution to the reaction; and (e) purifying the solution from (c) to obtain a final product, wherein said final product contains said bismuth-213-labeled compound.
 10. The method of claim 9, wherein said elution buffer comprises a NaI/HCl solution or a NaBr/HCl solution.
 11. The method of claim 10, wherein said elution buffer further comprises an antioxidant to prevent said elution buffer from oxidizing.
 12. The method of claim 11, wherein said antioxidant is l-ascorbic acid.
 13. The method of claim 9, wherein said compound is premixed with a neutralizing buffer.
 14. The method of claim 13, wherein said neutralizing buffer is selected from the group consisting of NH₄Ac, Na citrate and NH₄ citrate.
 15. The method of claim 9, wherein said reaction is run at a temperature higher than room temperature.
 16. The method of claim 9, wherein said quench solution is selected from the group consisting of EDTA, DTPA, EDTMP, EDTA+human serum albumin, DTPA+human serum albumin, EDTMP+human serum albumin and a chelate.
 17. The method of claim 9, wherein said method of purification is selected from the group consisting of size exclusion chromatography, ion exchange purification, reverse phase chromatography and affinity chromatography.
 18. The method of claim 9, wherein said compound is selected from the group consisting of an antibody, a fragment of an antibody, a cytokine and a receptor ligand.
 19. The method of claim 9, wherein processing time for completing said steps is from about 10 minutes to about 25 minutes.
 20. The method of claim 9, wherein said generator column reduces the leakage of ²²⁵Ac.
 21. The method of claim 9, wherein said generator column is capable of producing from about 10 mCi to about 100 mCi of Bismuth-213.
 22. A system for preparing a bismuth-213-labeled compound, comprising: a first container, a generator column loaded with actinium-225 by the generator of claim 1; a reaction vial; a second container; purification means; and a third container, said third container collecting a final product, wherein said final product contains said bismuth-213-labeled compound.
 23. The system of claim 22, wherein said purification means is a size exclusion column, an ion exchange column, a reverse phase column, an affinity column or a disc filter.
 24. The system of claim 22, wherein said first container, said second container and said third container are individually a syringe or a delivery device.
 25. The system of claim 22, wherein said first container contains an elution buffer.
 26. The system of claim 25, wherein said elution buffer comprises a NaI/HCl solution or a NaBr/HCl solution.
 27. The system of claim 26, wherein said elution buffer further comprises an antioxidant.
 28. The system of claim 27, wherein said antioxidant is l-ascorbic acid.
 29. The system of claim 22, wherein said reaction vial contains said compound premixed with a neutralizing buffer.
 30. The system of claim 29, wherein said neutralizing buffer is selected from the group consisting of NH₄Ac, Na citrate and NH₄ citrate.
 31. The system of claim 22, wherein said second container contains a quench solution.
 32. The system of claim 31, wherein said quench solution is selected from the group consisting of EDTA, DTPA, EDTMP, EDTA+human serum albumin, DTPA+human serum albumin, EDTMP+human serum albumin and a chelator.
 33. The method of claim 22, wherein said compound is selected from the group consisting of an antibody, a fragment of an antibody, a cytokine and a receptor ligand.
 34. A kit for preparing a bismuth-213-labeled compound, wherein said kit comprises: (a) a bismuth-2 13 generator, comprising: a first container containing ²²⁵Ac solution; a second container; a generator column; a valve, said valve simultaneously and directly connected to an end of each of said first container, said second container and said column; and a third container, said third container connected to said generator column at an exit end opposite to the end connected to said valve; and (b) a fourth container to contain an eluant; a reaction vial to contain said unlabeled compound premixed with a neutralizing buffer; a fifth container to contain a quenching solution; purification means; and a sixth container, said sixth container collecting a final product, wherein said final product contains said bismuth-213-labeled compound.
 35. The kit of claim 34, wherein said first container, said second container and said third container are individually a syringe, or a delivery device.
 36. The kit of claim 34, wherein said valve is a 3-way stopcock.
 37. The kit of claim 34, wherein said second container contains a resin and said third container applies a negative pressure for loading said resin onto said generator column.
 38. The kit of claim 37, wherein said resin is equilibrated with HCl solution. 